7-Phenyl-substituted tetracycline compounds

ABSTRACT

7-phenyl-substituted tetracycline compounds which are substantially free of positional isomers, methods of treating tetracycline responsive states, and pharmaceutical compositions containing the 7-phenyl-substituted tetracycline compounds are described. 7-substituted tetracycline compounds which are substantially free of positional isomers, methods of treating tetracycline responsive states, and pharmaceutical compositions containing the 7-substituted tetracycline compounds are described.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/853,536, filed May 24, 2004; which is a continuation of Ser. No.09/883,137, filed on Jun. 15, 2001; which claims priority to U.S.Provisional Application Ser. No. 60/212,030, filed on Jun. 16, 2000, andU.S. Provisional Application Ser. No. 60/212,471, filed on Jun. 16,2000; the entire contents of each of the aforementioned applications arehereby incorporated herein by reference.

BACKGROUND OF THE INVENTION

The development of the tetracycline antibiotics was the direct result ofa systematic screening of soil specimens collected from many parts ofthe world for evidence of microorganisms capable of producingbacteriocidal and/or bacteriostatic compositions. The first of thesenovel compounds was introduced in 1948 under the name chlortetracycline.Two years later, oxytetracycline became available. The elucidation ofthe chemical structure of these compounds confirmed their similarity andfurnished the analytical basis for the production of a third member ofthis group in 1952, tetracycline. A new family of tetracyclinecompounds, without the ring-attached methyl group present in earliertetracyclines, was prepared in 1957 and became publicly available in1967; and minocycline was in use by 1972.

Recently, research efforts have focused on developing new tetracyclineantibiotic compositions effective under varying therapeutic conditionsand routes of administration. New tetracycline analogues have also beeninvestigated which may prove to be equal to or more effective than theoriginally introduced tetracycline compounds. Examples include U.S. Pat.Nos. 3,957,980; 3,674,859; 2,980,584; 2,990,331; 3,062,717; 3,557,280;4,018,889; 4,024,272; 4,126,680; 3,454,697; and 3,165,531. These patentsare representative of the range of pharmaceutically active tetracyclineand tetracycline analogue compositions.

Historically, soon after their initial development and introduction, thetetracyclines were found to be highly effective pharmacologicallyagainst rickettsiae; a number of gram-positive and gram-negativebacteria; and the agents responsible for lymphogranuloma venereum,inclusion conjunctivitis, and psittacosis. Hence, tetracyclines becameknown as “broad spectrum” antibiotics. With the subsequent establishmentof their in vitro antimicrobial activity, effectiveness in experimentalinfections, and pharmacological properties, the tetracyclines as a classrapidly became widely used for therapeutic purposes. However, thiswidespread use of tetracyclines for both major and minor illnesses anddiseases led directly to the emergence of resistance to theseantibiotics even among highly susceptible bacterial species bothcommensal and pathogenic (e.g., pneumococci and Salmonella). The rise oftetracycline-resistant organisms has resulted in a general decline inuse of tetracyclines and tetracycline analogue compositions asantibiotics of choice.

SUMMARY OF THE INVENTION

The invention pertains to 7-substituted tetracycline compounds which aresubstantially free of positional isomers. The compounds have theformula:

wherein:

R⁴ and R^(4′) are each alkyl;

R⁵ is hydrogen, hydroxyl, or a prodrug moiety;

R⁶ and R^(6′) are each independently hydrogen, hydroxyl, alkyl, or takentogether, alkenyl;

R⁷ is halo substituted, a N-substituted phenyl, or an unsubstitutedphenyl; and pharmaceutically acceptable salts thereof.

In another embodiment, R⁷ is mono-, di-, or tri-halo substituted phenyl.In another embodiment, R⁷ is 2-halo substituted phenyl. In yet anotherembodiment, R⁷ is 3-halo substituted phenyl. In a further embodiment, R⁷is 4-halo substituted phenyl. In another embodiment, R⁷ is a 2, 3 or4-N-substituted phenyl.

A method is provided for preparing tetracycline compounds of theinvention having a desired purity, e.g., such that the desired compoundis provided in a manner substantially free of positional isomers. In apreferred embodiment, a 7-halo substituted sancycline compound, fromwhich the tetracycline compounds of the invention may be prepared, isprovided which enables the production of the desired compound in amanner substantially free of positional isomers. In a further preferredembodiment, the desired tetracycline compound is at least 75%, 80%, 85%,90%, 95% or 100% free of positional isomers. These desired compounds maybe used in methods of treating tetracycline responsive states, andpharmaceutical compositions, as substantially set forth herein.

The invention also pertains to a method for treating a tetracyclineresponsive state in a mammal, by administering to a mammal a compound offormula I substantially free of positional isomers. In another aspect,the invention relates to the use of a compound of formula I which issubstantially free of positional isomers to treat a tetracyclineresponsive state. The invention also pertains to pharmaceuticalcompositions comprising a compound of formula I which is substantiallyfree of positional isomers, and to the use of a compound of formula Iwhich is substantially free of positional isomers in the manufacture ofa medicament to treat a tetracycline responsive state.

DETAILED DESCRIPTION OF THE INVENTION

The invention pertains to 7-substituted tetracycline compounds which aresubstantially free of positional isomers. The compounds have theformula:

wherein:

R⁴ and R^(4′) are each alkyl;

R⁵ is hydrogen, hydroxyl, or a prodrug moiety;

R⁶ and R^(6′) are each independently hydrogen, hydroxyl, alkyl, or takentogether, alkenyl;

R⁷ is halo substituted, N-substituted phenyl or unsubstituted phenyl;and pharmaceutically acceptable salts thereof.

In another embodiment, R⁷ is mono-, di-, or tri-halo substituted phenyl.In another embodiment, R⁷ is 2-halo substituted phenyl. In yet anotherembodiment, R⁷ is 3-halo substituted phenyl. In a further embodiment, R⁷is 4-halo substituted phenyl.

In another further embodiment, R⁷ is a 2, 3 or 4-N-substituted phenyl,e.g., 7-(2-aminophenyl) sancycline, 7-(2-nitrophenyl) sancycline,7-(2-N,N,-dimethylaminophenyl) sancycline, 7-(2-N,N,-diethylaminophenyl)sancycline, 7-(2-N,N,-dipropylaminophenyl) sancycline,7-(2-N,N,-dibutylaminophenyl) sancycline, 7-(3-aminophenyl) sancycline,7-(3-nitrophenyl) sancycline, 7-(3-N,N,-dimethylaminophenyl) sancycline,7-(3-N,N,-diethylaminophenyl) sancycline, 7-(3-N,N,-dipropylaminophenyl)sancycline, 7-(3-N,N,-dibutylaminophenyl) sancycline, 7-(4-aminophenyl)sancycline, 7-(4-nitrophenyl) sancycline, 7-(4-N,N,-dimethylaminophenyl)sancycline, 7-(4-N,N,-diethylaminophenyl) sancycline,7-(4-N,N,-dipropylaminophenyl) sancycline, or7-(4-N,N,-dibutylaminophenyl) sancycline, where said compounds aresubstantially free of positional isomers.

The term “tetracycline compound” includes compounds with a similar ringstructure to tetracycline, such as those included in formula I. Someexamples of tetracycline compounds which can be modified to include asubstituent at position 7 include tetracycline, oxytetracycline,demeclocycline, methacycline, sancycline, and doxycycline; however,other derivatives and analogues comprising a similar ring structure arealso included. Table 1 depicts tetracycline and several knowntetracycline derivatives. TABLE I

The term “7-substituted tetracycline compounds” includes tetracyclinecompounds with a substituent at the 7 position. In an embodiment, thesubstituted tetracycline compound is substituted tetracycline (e.g.,wherein R⁴ and R^(4′) are methyl, R⁵ is hydrogen, R⁶ is methyl andR^(6′) is hydroxyl); substituted doxycycline (e.g., wherein R⁴ andR^(4′) are methyl, R⁵ is hydroxyl R⁶ is methyl and R^(6′) is hydrogen);or substituted sancycline (wherein R⁴ and R^(4′) are methyl; R⁵ ishydrogen and R⁶ and R^(6′) are hydrogen atoms). In another embodiment,the compound is a derivative of tetracycline, sancycline, doxycycline,oxytetracycline, or methacycline. In one embodiment, R⁵, R⁶ and R^(6′)are each hydrogen and R⁴ and R^(4′) are each methyl.

In yet another further embodiment, R⁷ is unsubstituted phenyl. Examplesof tetracycline compounds with this R⁷ substituent include 7-phenylsancycline and 7,9 diphenylsancycline, where said compounds aresubstantially free of positional isomers.

In yet another embodiment, R⁷ is halo substituted phenyl. The halosubstituent can be, for example, chlorine, fluorine, bromine, or iodine,as well as mono-, di- or tri-halo substituted lower alkyl group, e.g.,mono-, di- or tri-halo substituted methyl. In certain embodiments, thehalo substitution of the phenyl substituent enhances the ability of thetetracycline compound to perform its intended function, e.g., treattetracycline responsive states.

In a further embodiment, R⁷ is mono-, di-, or tri-halo substitutedphenyl. In certain embodiments, the 7-substituted tetracycline compoundis 7-(2,4-difluorophenyl) sancycline, 7-(2,4-dichlorophenyl) sancycline,7-(2,4-dibromophenyl) sancycline, or 7-(2,4-diiodophenyl) sancycline. Inother embodiments, the 7-substituted tetracycline compound is7-(2,6-difluorophenyl) sancycline, 7-(2,6-dichlorophenyl) sancycline,7-(2,6-dibromophenyl) sancycline, or 7-(2,6-diiodophenyl) sancycline,where said compounds are substantially free of positional isomers.

In another embodiment, R⁷ is 2-halo substituted phenyl, e.g.,7-(2-fluorophenyl) sancycline, 7-(2-chlorophenyl) sancycline,7-(2-bromophenyl) sancycline, or 7-(2-iodophenyl) sancycline, where saidcompounds are substantially free of positional isomers.

In yet another embodiment, R⁷ is 3-halo substituted phenyl, e.g.,7-(3-fluorophenyl) sancycline, 7-(3-chlorophenyl) sancycline,7-(3-bromophenyl) sancycline, or 7-(3-iodophenyl) sancycline, where saidcompounds are substantially free of positional isomers.

In further embodiment, R⁷ is 4-halo substituted phenyl, e.g.,7-(4-fluorophenyl) sancycline, 7-(4-chlorophenyl) sancycline,7-(4-bromophenyl) sancycline, or 7-(4-iodophenyl) sancycline, where saidcompounds are substantially free of positional isomers.

In yet another embodiment, R⁷ is a mono-, di-, or tri-substituted phenylwhere the substituent is a mono-, di- or tri-halo substituted loweralkyl group, e.g., mono-, di- or tri-halo substituted methyl. Forexample, the compound may be 7-(4-trichloromethylphenyl) sancycline,7-(4-trifluoromethylphenyl) sancycline, 7-(4-tribromomethylphenyl)sancycline, or 7-(4-triiodomethylphenyl) sancycline, where saidcompounds are substantially free of positional isomers.

The term “N-substituted phenyl” includes phenyl substituted with one ormore substituents with a nitrogen atom such as, but not limited to,nitro, amino, alkyl amino, and dialkylamino (e.g., dimethyl amino,diethylamino, dipropyl amino, dibutyl amino). The N-substituent may beat the 2, 3, 4, 5, or 6 position of the phenyl ring.

In another embodiment, R⁷ is a 2-N-substituted phenyl, including7-(2-aminophenyl) sancycline, 7-(2-nitrophenyl) sancycline,7-(2-N,N,-dimethylaminophenyl) sancycline, 7-(2-N,N,-diethylaminophenyl)sancycline, 7-(2-N,N,-dipropylaminophenyl) sancycline, and7-(2-N,N,-dibutylaminophenyl) sancycline, where said compounds aresubstantially free of positional isomers.

In another embodiment, R⁷ is a 3-N-substituted phenyl, including7-(3-aminophenyl) sancycline, 7-(3-nitrophenyl) sancycline,7-(3-N,N,-dimethylaminophenyl) sancycline, 7-(3-N,N,-diethylaminophenyl)sancycline, 7-(3-N,N,-dipropylaminophenyl) sancycline, and7-(3-N,N,-dibutylaminophenyl) sancycline, where said compounds aresubstantially free of positional isomers.

In another embodiment, R⁷ is 4-N-substituted phenyl, including7-(4-aminophenyl) sancycline, 7-(4-nitrophenyl) sancycline,7-(4-N,N,-dimethylaminophenyl) sancycline, 7-(4-N,N,-diethylaminophenyl)sancycline, 7-(4-N,N,-dipropylaminophenyl) sancycline, and7-(4-N,N,-dibutylaminophenyl) sancycline, where said compounds aresubstantially free of positional isomers.

The 7-substituted compounds of the invention can be synthesized bymethods known in the art and/or as described herein. In Scheme 1, ageneral synthetic scheme is outlined using a Suzuki coupling of aboronic acid with an iodo sancycline compound. Although the reaction isshown for sancycline, a similar procedure can be used for othertetracycline compounds. Furthermore, other aryl coupling reactions knownin the art may also be used.

As shown in Scheme 1, an iodosancycline compound can be synthesized fromunsubstituted sancycline by treating it with at least one equivalentN-iodosuccinimide (NIS) under acidic conditions. When sancycline wastreated with NIS in trifluoroacetic acid, the reaction was carried outinitially at 0° C., before being warmed to room temperature for fivehours. The reaction is then quenched, and the resulting 7-iodosancyclinecan then be purified using standard techniques known in the art. The7-iodosancycline can then be further reacted with a boronic acid, asshown in Scheme 1. 7-iodosancycline, a palladium catalyst (such asPd(OAc)₂), is dissolved in a solvent and treated with aqueous sodiumcarbonate, and the boronic acid. The resulting compound can then bepurified using techniques known in the art such as preparative HPLC andcharacterized.

The compounds of the invention can also be synthesized using Stillecross couplings. Stille cross couplings can be performed using anappropriate tin reagent (e.g., R—SnBu₃) and a halogenated tetracyclinecompound, (e.g., 7-iodosancycline). The tin reagent and theiodotetracycline compound can be treated with a palladium catalyst(e.g., Pd(PPh₃)₂Cl₂ or Pd(AsPh₃)₂Cl₂) and, optionally, with anadditional copper salt, e.g., CuI. The resulting compound can then bepurified using techniques known in the art. The synthesis of thecompounds of the invention are described in more detail in Example 1.

The language “a compound which is substantially free of positionalisomers” includes a compound which has been isolated and/or purifiedfrom its positional isomers such that the desired compound is present inan amount acceptable for pharmaceutical purposes, e.g., at least 75percent. The percent purity can be determined using art-recognizedtechniques. As set forth in exemplary fashion below, a method isprovided for preparing tetracycline compounds of the invention having adesired purity, e.g., such that the desired compound is provided in amanner substantially free of positional isomers. In a preferredembodiment, a 7-halo substituted sancycline compound, from which thetetracycline compounds of the invention may be prepared, is providedwhich enables the production of the desired compound in a mannersubstantially free of positional isomers. In a further preferredembodiment, the desired tetracycline compound thus produced is at least75%, 80%, 85%, 90%, 95% or 100% free of positional isomers.

The term “alkyl” includes saturated aliphatic groups, includingstraight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chain alkyl groups(isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups(cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkylsubstituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.The term alkyl further includes alkyl groups, which comprise oxygen,nitrogen, sulfur or phosphorous atoms replacing one or more carbons ofthe hydrocarbon backbone. In certain embodiments, a straight chain orbranched chain alkyl has 6 or fewer carbon atoms in its backbone (e.g.,C₁-C₆ for straight chain, C₃-C₆ for branched chain), and more preferably4 or fewer. Likewise, preferred cycloalkyls have from 3-8 carbon atomsin their ring structure, and more preferably have 5 or 6 carbons in thering structure. The term C₁-C₆ includes alkyl groups containing 1 to 6carbon atoms.

Moreover, the term alkyl includes both “unsubstituted alkyls” and“substituted alkyls”, the latter of which refers to alkyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example, alkyl,alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkylamino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety. Cycloalkyls can be further substituted, e.g.,with the substituents described above. An “alkylaryl” or an “aralkyl”moiety is an alkyl substituted with an aryl (e.g., phenylmethyl(benzyl)). The term “alkyl” also includes the side chains of natural andunnatural amino acids.

The term “aryl” includes groups with aromaticity, including 5- and6-membered single-ring aromatic groups that may include from zero tofour heteroatoms as well as multicyclic systems with at least onearomatic ring. Examples of aryl groups include benzene, phenyl, pyrrole,furan, thiophene, thiazole, isothiazole, imidazole, triazole, tetrazole,pyrazole, oxazole, isooxazole, pyridine, pyrazine, pyridazine, andpyrimidine, and the like. Furthermore, the term “aryl” includesmulticyclic aryl groups, e.g., tricyclic, bicyclic, e.g., naphthalene,benzoxazole, benzodioxazole, benzothiazole, benzoimidazole,benzothiophene, methylenedioxyphenyl, quinoline, isoquinoline,napthridine, indole, benzofuran, purine, benzofuran, deazapurine, orindolizine. Those aryl groups having heteroatoms in the ring structuremay also be referred to as “aryl heterocycles”, “heterocycles,”“heteroaryls” or “heteroaromatics”. The aromatic ring can be substitutedat one or more ring positions with such substituents as described above,as for example, halogen, hydroxyl, alkoxy, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, alkylaminocarbonyl, aralkylaminocarbonyl,alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl,alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety. Aryl groups can also be fused or bridged withalicyclic or heterocyclic rings which are not aromatic so as to form amulticyclic system (e.g., tetralin, methylenedioxyphenyl).

The term “alkenyl” includes unsaturated aliphatic groups analogous inlength and possible substitution to the alkyls described above, but thatcontain at least one double bond.

For example, the term “alkenyl” includes straight-chain alkenyl groups(e.g., ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl,nonenyl, decenyl, etc.), branched-chain alkenyl groups, cycloalkenyl(alicyclic) groups (cyclopropenyl, cyclopentenyl, cyclohexenyl,cycloheptenyl, cyclooctenyl), alkyl or alkenyl substituted cycloalkenylgroups, and cycloalkyl or cycloalkenyl substituted alkenyl groups. Theterm alkenyl further includes alkenyl groups which include oxygen,nitrogen, sulfur or phosphorous atoms replacing one or more carbons ofthe hydrocarbon backbone. In certain embodiments, a straight chain orbranched chain alkenyl group has 6 or fewer carbon atoms in its backbone(e.g., C₂-C₆ for straight chain, C₃-C₆ for branched chain). Likewise,cycloalkenyl groups may have from 3-8 carbon atoms in their ringstructure, and more preferably have 5 or 6 carbons in the ringstructure. The term C₂-C₆ includes alkenyl groups containing 2 to 6carbon atoms.

Moreover, the term alkenyl includes both “unsubstituted alkenyls” and“substituted alkenyls”, the latter of which refers to alkenyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example, alkylgroups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety.

The term “alkynyl” includes unsaturated aliphatic groups analogous inlength and possible substitution to the alkyls described above, butwhich contain at least one triple bond.

For example, the term “alkynyl” includes straight-chain alkynyl groups(e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl,nonynyl, decynyl, etc.), branched-chain alkynyl groups, and cycloalkylor cycloalkenyl substituted alkynyl groups. The term alkynyl furtherincludes alkynyl groups which include oxygen, nitrogen, sulfur orphosphorous atoms replacing one or more carbons of the hydrocarbonbackbone. In certain embodiments, a straight chain or branched chainalkynyl group has 6 or fewer carbon atoms in its backbone (e.g., C₂-C₆for straight chain, C₃-C₆ for branched chain). The term C₂-C₆ includesalkynyl groups containing 2 to 6 carbon atoms.

Moreover, the term alkynyl includes both “unsubstituted alkynyls” and“substituted alkynyls”, the latter of which refers to alkynyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example, alkylgroups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto five carbon atoms in its backbone structure. “Lower alkenyl” and“lower alkynyl” have chain lengths of, for example, 2-5 carbon atoms.

The term “acyl” includes compounds and moieties which contain the acylradical (CH₃CO—) or a carbonyl group. The term “substituted acyl”includes acyl groups where one or more of the hydrogen atoms arereplaced by for example, alkyl groups, alkynyl groups, halogens,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano,amino (including alkylamino, dialkylamino, arylamino, diarylamino, andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

The term “acylamino” includes moieties wherein an acyl moiety is bondedto an amino group. For example, the term includes alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido groups.

The term “aroyl” includes compounds and moieties with an aryl orheteroaromatic moiety bound to a carbonyl group. Examples of aroylgroups include phenylcarboxy, naphthyl carboxy, etc.

The terms “alkoxyalkyl”, “alkylaminoalkyl” and “thioalkoxyalkyl” includealkyl groups, as described above, which further include oxygen, nitrogenor sulfur atoms replacing one or more carbons of the hydrocarbonbackbone, e.g., oxygen, nitrogen or sulfur atoms.

The term “alkoxy” includes substituted and unsubstituted alkyl, alkenyl,and alkynyl groups covalently linked to an oxygen atom. Examples ofalkoxy groups include methoxy, ethoxy, isopropyloxy, propoxy, butoxy,and pentoxy groups. Examples of substituted alkoxy groups includehalogenated alkoxy groups. The alkoxy groups can be substituted withgroups such as alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moieties. Examples of halogen substituted alkoxygroups include, but are not limited to, fluoromethoxy, difluoromethoxy,trifluoromethoxy, chloromethoxy, dichloromethoxy, trichloromethoxy, etc.

The term “amine” or “amino” includes compounds where a nitrogen atom iscovalently bonded to at least one carbon or heteroatom. The term“alkylamino” includes groups and compounds wherein the nitrogen is boundto at least one additional alkyl group. The term “dialkylamino” includesgroups wherein the nitrogen atom is bound to at least two additionalalkyl groups. The term “arylamino” and “diarylamino” include groupswherein the nitrogen is bound to at least one or two aryl groups,respectively. The term “alkylarylamino,” “alkylaminoaryl” or“arylaminoalkyl” refers to an amino group which is bound to at least onealkyl group and at least one aryl group. The term “alkaminoalkyl” refersto an alkyl, alkenyl, or alkynyl group bound to a nitrogen atom which isalso bound to an alkyl group.

The term “amide” or “aminocarboxy” includes compounds or moieties whichcontain a nitrogen atom which is bound to the carbon of a carbonyl or athiocarbonyl group. The term includes “alkaminocarboxy” groups whichinclude alkyl, alkenyl, or alkynyl groups bound to an amino group boundto a carboxy group. It includes arylaminocarboxy groups which includearyl or heteroaryl moieties bound to an amino group which is bound tothe carbon of a carbonyl or thiocarbonyl group. The terms“alkylaminocarboxy,” “alkenylaminocarboxy,” “alkynylaminocarboxy,” and“arylaminocarboxy” include moieties wherein alkyl, alkenyl, alkynyl andaryl moieties, respectively, are bound to a nitrogen atom which is inturn bound to the carbon of a carbonyl group.

The term “carbonyl” or “carboxy” includes compounds and moieties whichcontain a carbon connected with a double bond to an oxygen atom.Examples of moieties which contain a carbonyl include aldehydes,ketones, carboxylic acids, amides, esters, anhydrides, etc.

The term “thiocarbonyl” or “thiocarboxy” includes compounds and moietieswhich contain a carbon connected with a double bond to a sulfur atom.

The term “ether” includes compounds or moieties which contain an oxygenbonded to two different carbon atoms or heteroatoms. For example, theterm includes “alkoxyalkyl” which refers to an alkyl, alkenyl, oralkynyl group covalently bonded to an oxygen atom which is covalentlybonded to another alkyl group.

The term “ester” includes compounds and moieties which contain a carbonor a heteroatom bound to an oxygen atom which is bonded to the carbon ofa carbonyl group. The term “ester” includes alkoxycarboxy groups such asmethoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl,pentoxycarbonyl, etc. The alkyl, alkenyl, or alkynyl groups are asdefined above.

The term “thioether” includes compounds and moieties which contain asulfur atom bonded to two different carbon or hetero atoms. Examples ofthioethers include, but are not limited to alkthioalkyls,alkthioalkenyls, and alkthioalkynyls. The term “alkthioalkyls” includecompounds with an alkyl, alkenyl, or alkynyl group bonded to a sulfuratom which is bonded to an alkyl group. Similarly, the term“alkthioalkenyls” and alkthioalkynyls” refer to compounds or moietieswherein an alkyl, alkenyl, or alkynyl group is bonded to a sulfur atomwhich is covalently bonded to an alkynyl group.

The term “hydroxy” or “hydroxyl” includes groups with an —OH or —O⁻.

The term “halogen” includes fluorine, bromine, chlorine, iodine, etc.The term “perhalogenated” generally refers to a moiety wherein allhydrogens are replaced by halogen atoms.

The terms “polycyclyl” or “polycyclic radical” refer to two or morecyclic rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, arylsand/or heterocyclyls) in which two or more carbons are common to twoadjoining rings. Rings that are joined through non-adjacent atoms aretermed “bridged” rings. Each of the rings of the polycycle can besubstituted with such substituents as described above, as for example,halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl,alkylaminocarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl,alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl,aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato,phosphinato, cyano, amino (including alkylamino, dialkylamino,arylamino, diarylamino, and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkyl, alkylaryl, or anaromatic or heteroaromatic moiety.

The term “heteroatom” includes atoms of any element other than carbon orhydrogen. Examples of heteroatoms include nitrogen, oxygen, sulfur andphosphorus.

It will be noted that the structure of some of the compounds of thisinvention includes asymmetric carbon atoms. It is to be understoodaccordingly that the isomers arising from such asymmetry (e.g., allenantiomers and diastereomers) are included within the scope of thisinvention, unless indicated otherwise. Such isomers can be obtained insubstantially pure form by classical separation techniques and bystereochemically controlled synthesis. Furthermore, the structures andother compounds and moieties discussed in this application also includeall tautomers thereof.

Prodrugs are compounds which are converted in vivo to active forms (see,e.g., R. B. Silverman, 1992, “The Organic Chemistry of Drug Design andDrug Action”, Academic Press, Chp. 8). Prodrugs can be used to alter thebiodistribution (e.g., to allow compounds which would not typicallyenter the reactive site of the protease) or the pharmacokinetics for aparticular compound. For example, a hydroxyl group, can be esterified,e.g., with a carboxylic acid group to yield an ester. When the ester isadministered to a subject, the ester is cleaved, enzymatically ornon-enzymatically, reductively or hydrolytically, to reveal the hydroxylgroup.

The term “prodrug moiety” includes moieties which can be metabolized invivo to a hydroxyl group and moieties which may advantageously remainesterified in vivo. Preferably, the prodrugs moieties are metabolized invivo by esterases or by other mechanisms to hydroxyl groups or otheradvantageous groups. Examples of prodrugs and their uses are well knownin the art (See, e.g., Berge et al. (1977) “Pharmaceutical Salts”, J.Pharm. Sci. 66:1-19). The prodrugs can be prepared in situ during thefinal isolation and purification of the compounds, or by separatelyreacting the purified compound in its free acid form or hydroxyl with asuitable esterifying agent. Hydroxyl groups can be converted into estersvia treatment with a carboxylic acid. Examples of prodrug moietiesinclude substituted and unsubstituted, branch or unbranched lower alkylester moieties, (e.g., propionoic acid esters), lower alkenyl esters,di-lower alkyl-amino lower-alkyl esters (e.g., dimethylaminoethylester), acylamino lower alkyl esters (e.g., acetyloxymethyl ester),acyloxy lower alkyl esters (e.g., pivaloyloxymethyl ester), aryl esters(phenyl ester), aryl-lower alkyl esters (e.g., benzyl ester),substituted (e.g., with methyl, halo, or methoxy substituents) aryl andaryl-lower alkyl esters, amides, lower-alkyl amides, di-lower alkylamides, and hydroxy amides. Preferred prodrug moieties are propionoicacid esters and acyl esters.

The invention also features a method for treating a tetracyclinecompound responsive state in a subject, by administering to the subjecta 7-substituted tetracycline compound of the invention, e.g., a compoundof formula I which is substantially free of positional isomers.Preferably, an effective amount of the tetracycline compound which issubstantially free of positional isomers is administered. Examples of7-substituted tetracycline compounds of the invention include 7-phenylsancycline, 7,9 diphenylsancycline, 7-(2-fluorophenyl) sancycline,7-(2-chlorophenyl) sancycline, 7-(2-bromophenyl) sancycline,7-(2-iodophenyl) sancycline, 7-(3-fluorophenyl) sancycline,7-(3-chlorophenyl) sancycline, 7-(3-bromophenyl) sancycline,7-(3-iodophenyl) sancycline, 7-(4-fluorophenyl) sancycline,7-(4-chlorophenyl) sancycline, 7-(4-bromophenyl) sancycline,7-(4-iodophenyl) sancycline, 7-(4-trichloromethylphenyl) sancycline,7-(4-trifluoromethylphenyl) sancycline, 7-(4-tribromomethylphenyl)sancycline, 7-(4-triiodomethylphenyl) sancycline, 7-(2-aminophenyl)sancycline, 7-(2-nitrophenyl) sancycline, 7-(2-N,N,-dimethylaminophenyl)sancycline, 7-(2-N,N,-diethylaminophenyl) sancycline,7-(2-N,N,-dipropylaminophenyl) sancycline, 7-(2-N,N,-dibutylaminophenyl)sancycline, 7-(3-aminophenyl) sancycline, 7-(3-nitrophenyl) sancycline,7-(3-N,N,-dimethylaminophenyl) sancycline, 7-(3-N,N,-diethylaminophenyl)sancycline, 7-(3-N,N,-dipropylaminophenyl) sancycline,7-(3-N,N,-dibutylaminophenyl) sancycline, 7-(4-aminophenyl) sancycline,7-(4-nitrophenyl) sancycline, 7-(4-N,N,-dimethylaminophenyl) sancycline,7-(4-N,N,-diethylaminophenyl) sancycline, 7-(4-N,N,-dipropylaminophenyl)sancycline, and 7-(4-N,N,-dibutylaminophenyl) sancycline, where saidcompounds are substantially free of positional isomers.

The language “tetracycline compound responsive state” includes stateswhich can be treated, prevented, or otherwise ameliorated by theadministration of a tetracycline compound of the invention. Tetracyclinecompound responsive states include bacterial infections (including thosewhich are resistant to other tetracycline compounds), cancer, diabetes,and other states for which tetracycline compounds have been found to beactive (see, for example, U.S. Pat. Nos. 5,789,395; 5,834,450; and5,532,227). Compounds of the invention can be used to prevent or controlimportant mammalian and veterinary diseases such as diarrhea, urinarytract infections, infections of skin and skin structure, ear, nose andthroat infections, wound infection, mastitis and the like. In addition,methods for treating neoplasms using tetracycline compounds of theinvention are also included (van der Bozert et al., Cancer Res.,48:6686-6690 (1988)).

Bacterial infections may be caused by a wide variety of gram positiveand gram negative bacteria. The compounds of the invention are useful asantibiotics against organisms which are resistant to other tetracyclinecompounds. The antibiotic activity of the tetracycline compounds of theinvention may be determined using the method discussed in Example 2, orby using the in vitro standard broth dilution method described in Waitz,J. A., National Commission for Clinical Laboratory Standards, DocumentM7-A2, vol. 10, no. 8, pp. 13-20, 2^(nd) edition, Villanova, Pa. (1990).

The tetracycline compounds may also be used to treat infectionstraditionally treated with tetracycline compounds such as, for example,rickettsiae; a number of gram-positive and gram-negative bacteria; andthe agents responsible for lymphogranuloma venereum, inclusionconjunctivitis, psittacosis. The tetracycline compounds may be used totreat infections of, e.g., K. pneumoniae, Salmonella, E. hirae, A.baumanii, B. catarrhalis, H. influenzae, P. aeruginosa, E. faecium, E.coli, S. aureus or E. faecalis. In one embodiment, the tetracyclinecompound is used to treat a bacterial infection that is resistant toother tetracycline antibiotic compounds. The tetracycline compound ofthe invention may be administered with a pharmaceutically acceptablecarrier.

The language “effective amount” of the compound is that amount necessaryor sufficient to treat or prevent a tetracycline compound responsivestate. The effective amount can vary depending on such factors as thesize and weight of the subject, the type of illness, or the particulartetracycline compound. For example, the choice of the tetracyclinecompound can affect what constitutes an “effective amount”. One ofordinary skill in the art would be able to study the aforementionedfactors and make the determination regarding the effective amount of thetetracycline compound without undue experimentation.

The invention also pertains to methods of treatment againstmicroorganism infections and associated diseases. The methods includeadministration of an effective amount of one or more tetracyclinecompounds to a subject. The subject can be either a plant or,advantageously, an animal, e.g., a mammal, e.g., a human.

In the therapeutic methods of the invention, one or more tetracyclinecompounds of the invention may be administered alone to a subject, ormore typically a compound of the invention will be administered as partof a pharmaceutical composition in mixture with conventional excipient,i.e., pharmaceutically acceptable organic or inorganic carriersubstances suitable for parenteral, oral or other desired administrationand which do not deleteriously react with the active compounds and arenot deleterious to the recipient thereof.

In one embodiment, the pharmaceutical composition comprises a7-substituted tetracycline compound of the invention, e.g., of formulaI. In a further embodiment, the 7-substituted tetracycline compound is7-phenyl sancycline, 7,9 diphenylsancycline, 7-(2-fluorophenyl)sancycline, 7-(2-chlorophenyl) sancycline, 7-(2-bromophenyl) sancycline,7-(2-iodophenyl) sancycline, 7-(3-fluorophenyl) sancycline,7-(3-chlorophenyl) sancycline, 7-(3-bromophenyl) sancycline,7-(3-iodophenyl) sancycline, 7-(4-fluorophenyl) sancycline,7-(4-chlorophenyl) sancycline, 7-(4-bromophenyl) sancycline,7-(4-iodophenyl) sancycline, 7-(4-trichloromethylphenyl) sancycline,7-(4-trifluoromethylphenyl) sancycline, 7-(4-tribromomethylphenyl)sancycline, or 7-(4-triiodomethylphenyl) sancycline, 7-(2-aminophenyl)sancycline, 7-(2-nitrophenyl) sancycline, 7-(2-N,N,-dimethylaminophenyl)sancycline, 7-(2-N,N,-diethylaminophenyl) sancycline,7-(2-N,N,-dipropylaminophenyl) sancycline, 7-(2-N,N,-dibutylaminophenyl)sancycline, 7-(3-aminophenyl) sancycline, 7-(3-nitrophenyl) sancycline,7-(3-N,N,-dimethylaminophenyl) sancycline, 7-(3-N,N,-diethylaminophenyl)sancycline, 7-(3-N,N,-dipropylaminophenyl) sancycline,7-(3-N,N-dibutylaminophenyl) sancycline, 7-(4-aminophenyl) sancycline,7-(4-nitrophenyl) sancycline, 7-(4-N,N,-dimethylaminophenyl) sancycline,7-(4-N,N,-diethylaminophenyl) sancycline, 7-(4-N,N,-dipropylaminophenyl)sancycline, and 7-(4-N,N,-dibutylaminophenyl) sancycline, where saidcompounds are substantially free of positional isomers.

The language “pharmaceutically acceptable carrier” includes substancescapable of being coadministered with the tetracycline compound(s), andwhich allow both to perform their intended function, e.g., treat orprevent a tetracycline compound responsive state. Suitablepharmaceutically acceptable carriers include but are not limited towater, salt solutions, alcohol, vegetable oils, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, perfume oil, fatty acid monoglycerides anddiglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose,polyvinylpyrrolidone, etc. The pharmaceutical preparations can besterilized and if desired mixed with auxiliary agents, e.g., lubricants,preservatives, stabilizers, wetting agents, emulsifiers, salts forinfluencing osmotic pressure, buffers, colorings, flavorings and/oraromatic substances and the like which do not deleteriously react withthe active compounds of the invention.

The tetracycline compounds of the invention that are basic in nature arecapable of forming a wide variety of salts with various inorganic andorganic acids. The acids that may be used to prepare pharmaceuticallyacceptable acid addition salts of the tetracycline compounds of theinvention that are basic in nature are those that form non-toxic acidaddition salts, i.e., salts containing pharmaceutically acceptableanions, such as the hydrochloride, hydrobromide, hydroiodide, nitrate,sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate,lactate, salicylate, citrate, acid citrate, tartrate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucaronate, saccharate, formate, benzoate, glutamate,methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonateand palmoate [i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)] salts.Although such salts must be pharmaceutically acceptable foradministration to a subject, e.g., a mammal, it is often desirable inpractice to initially isolate a tetracycline compound of the inventionfrom the reaction mixture as a pharmaceutically unacceptable salt andthen simply convert the latter back to the free base compound bytreatment with an alkaline reagent and subsequently convert the latterfree base to a pharmaceutically acceptable acid addition salt. The acidaddition salts of the base compounds of this invention are readilyprepared by treating the base compound with a substantially equivalentamount of the chosen mineral or organic acid in an aqueous solventmedium or in a suitable organic solvent, such as methanol or ethanol.Upon careful evaporation of the solvent, the desired solid salt isreadily obtained. The preparation of other tetracycline compounds of theinvention not specifically described in the foregoing experimentalsection can be accomplished using combinations of the reactionsdescribed above that will be apparent to those skilled in the art.

The preparation of other tetracycline compounds of the invention notspecifically described in the foregoing experimental section can beaccomplished using combinations of the reactions described above thatwill be apparent to those skilled in the art.

The tetracycline compounds of the invention that are acidic in natureare capable of forming a wide variety of base salts. The chemical basesthat may be used as reagents to prepare pharmaceutically acceptable basesalts of those tetracycline compounds of the invention that are acidicin nature are those that form non-toxic base salts with such compounds.Such non-toxic base salts include, but are not limited to those derivedfrom such pharmaceutically acceptable cations such as alkali metalcations (e.g., potassium and sodium) and alkaline earth metal cations(e.g., calcium and magnesium), ammonium or water-soluble amine additionsalts such as N-methylglucamine-(meglumine), and the loweralkanolammonium and other base salts of pharmaceutically acceptableorganic amines. The pharmaceutically acceptable base addition salts oftetracycline compounds of the invention that are acidic in nature may beformed with pharmaceutically acceptable cations by conventional methods.Thus, these salts may be readily prepared by treating the tetracyclinecompound of the invention with an aqueous solution of the desiredpharmaceutically acceptable cation and evaporating the resultingsolution to dryness, preferably under reduced pressure. Alternatively, alower alkyl alcohol solution of the tetracycline compound of theinvention may be mixed with an alkoxide of the desired metal and thesolution subsequently evaporated to dryness.

The preparation of other tetracycline compounds of the invention notspecifically described in the foregoing experimental section can beaccomplished using combinations of the reactions described above thatwill be apparent to those skilled in the art.

The tetracycline compounds of the invention and pharmaceuticallyacceptable salts thereof can be administered via either the oral,parenteral or topical routes. In general, these compounds are mostdesirably administered in effective dosages, depending upon the weightand condition of the subject being treated and the particular route ofadministration chosen. Variations may occur depending upon the speciesof the subject being treated and its individual response to saidmedicament, as well as on the type of pharmaceutical formulation chosenand the time period and interval at which such administration is carriedout.

The pharmaceutical compositions of the invention may be administeredalone or in combination with other known compositions for treatingtetracycline responsive states in a mammal. Preferred mammals includepets (e.g., cats, dogs, ferrets, etc.), farm animals (cows, sheep, pigs,horses, goats, etc.), lab animals (rats, mice, monkeys, etc.), andprimates (chimpanzees, humans, gorillas). The language “in combinationwith” a known composition is intended to include simultaneousadministration of the composition of the invention and the knowncomposition, administration of the composition of the invention first,followed by the known composition and administration of the knowncomposition first, followed by the composition of the invention. Any ofthe therapeutically composition known in the art for treatingtetracycline responsive states can be used in the methods of theinvention.

The compounds of the invention may be administered alone or incombination with pharmaceutically acceptable carriers or diluents by anyof the routes previously mentioned, and the administration may becarried out in single or multiple doses. For example, the noveltherapeutic agents of this invention can be administered advantageouslyin a wide variety of different dosage forms, i.e., they may be combinedwith various pharmaceutically acceptable inert carriers in the form oftablets, capsules, lozenges, troches, hard candies, powders, sprays,creams, salves, suppositories, jellies, gels, pastes, lotions,ointments, aqueous suspensions, injectable solutions, elixirs, syrups,and the like. Such carriers include solid diluents or fillers, sterileaqueous media and various non-toxic organic solvents, etc. Moreover,oral pharmaceutical compositions can be suitably sweetened and/orflavored. In general, the therapeutically-effective compounds of thisinvention are present in such dosage forms at concentration levelsranging from about 5.0% to about 70% by weight.

For oral administration, tablets containing various excipients such asmicrocrystalline cellulose, sodium citrate, calcium carbonate, dicalciumphosphate and glycine may be employed along with various disintegrantssuch as starch (and preferably corn, potato or tapioca starch), alginicacid and certain complex silicates, together with granulation binderslike polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally,lubricating agents such as magnesium stearate, sodium lauryl sulfate andtalc are often very useful for tabletting purposes. Solid compositionsof a similar type may also be employed as fillers in gelatin capsules;preferred materials in this connection also include lactose or milksugar as well as high molecular weight polyethylene glycols. Whenaqueous suspensions and/or elixirs are desired for oral administration,the active ingredient may be combined with various sweetening orflavoring agents, coloring matter or dyes, and, if so desired,emulsifying and/or suspending agents as well, together with suchdiluents as water, ethanol, propylene glycol, glycerin and various likecombinations thereof.

For parenteral administration (including intraperitoneal, subcutaneous,intravenous, intradermal or intramuscular injection), solutions of atherapeutic compound of the present invention in either sesame or peanutoil or in aqueous propylene glycol may be employed. The aqueoussolutions should be suitably buffered (preferably pH greater than 8) ifnecessary and the liquid diluent first rendered isotonic. These aqueoussolutions are suitable for intravenous injection purposes. The oilysolutions are suitable for intraarticular, intramuscular andsubcutaneous injection purposes. The preparation of all these solutionsunder sterile conditions is readily accomplished by standardpharmaceutical techniques well known to those skilled in the art. Forparenteral application, examples of suitable preparations includesolutions, preferably oily or aqueous solutions as well as suspensions,emulsions, or implants, including suppositories. Therapeutic compoundsmay be formulated in sterile form in multiple or single dose formatssuch as being dispersed in a fluid carrier such as sterile physiologicalsaline or 5% saline dextrose solutions commonly used with injectables.

Additionally, it is also possible to administer the compounds of thepresent invention topically when treating inflammatory conditions of theskin. Examples of methods of topical administration include transdermal,buccal or sublingual application. For topical applications, therapeuticcompounds can be suitably admixed in a pharmacologically inert topicalcarrier such as a gel, an ointment, a lotion or a cream. Such topicalcarriers include water, glycerol, alcohol, propylene-glycol, fattyalcohols, triglycerides, fatty acid esters, or mineral oils. Otherpossible topical carriers are liquid petrolatum, isopropylpalmitate,polyethylene glycol, ethanol 95%, polyoxyethylene monolauriate 5% inwater, sodium lauryl sulfate 5% in water, and the like. In addition,materials such as anti-oxidants, humectants, viscosity stabilizers andthe like also may be added if desired.

For enteral application, particularly suitable are tablets, dragees orcapsules having talc and/or carbohydrate carrier binder or the like, thecarrier preferably being lactose and/or corn starch and/or potatostarch. A syrup, elixir or the like can be used wherein a sweetenedvehicle is employed. Sustained release compositions can be formulatedincluding those wherein the active component is protected withdifferentially degradable coatings, e.g., by microencapsulation,multiple coatings, etc.

In addition to treatment of human subjects, the therapeutic methods ofthe invention also will have significant veterinary applications, e.g.for treatment of livestock such as cattle, sheep, goats, cows, swine andthe like; poultry such as chickens, ducks, geese, turkeys and the like;horses; and pets such as dogs and cats. Also, the compounds of theinvention may be used to treat non-animal subjects, such as plants.

It will be appreciated that the actual preferred amounts of activecompounds used in a given therapy will vary according to the specificcompound being utilized, the particular compositions formulated, themode of application, the particular site of administration, etc. Optimaladministration rates for a given protocol of administration can bereadily ascertained by those skilled in the art using conventionaldosage determination tests conducted with regard to the foregoingguidelines.

In general, compounds of the invention for treatment can be administeredto a subject in dosages used in prior tetracycline therapies. See, forexample, the Physicians' Desk Reference. For example, a suitableeffective dose of one or more compounds of the invention will be in therange of from 0.01 to 100 milligrams per kilogram of body weight ofrecipient per day, preferably in the range of from 0.1 to 50 milligramsper kilogram body weight of recipient per day, more preferably in therange of 1 to 20 milligrams per kilogram body weight of recipient perday. The desired dose is suitably administered once daily, or severalsub-doses, e.g. 2 to 5 sub-doses, are administered at appropriateintervals through the day, or other appropriate schedule.

It will also be understood that normal, conventionally known precautionswill be taken regarding the administration of tetracyclines generally toensure their efficacy under normal use circumstances. Especially whenemployed for therapeutic treatment of humans and animals in vivo, thepractitioner should take all sensible precautions to avoidconventionally known contradictions and toxic effects. Thus, theconventionally recognized adverse reactions of gastrointestinal distressand inflammations, the renal toxicity, hypersensitivity reactions,changes in blood, and impairment of absorption through aluminum,calcium, and magnesium ions should be duly considered in theconventional manner.

Furthermore, the invention also pertains to the use of a tetracyclinecompound of formula I which is substantially free of positional isomers,for the preparation of a medicament. The medicament may include apharmaceutically acceptable carrier and the tetracycline compound is aneffective amount, e.g., an effective amount to treat a tetracyclineresponsive state.

In yet another embodiment, the invention also pertains to the use of atetracycline compound of formula I which is substantially free ofpositional isomers to treat a tetracycline responsive state, e.g., in asubject, e.g., a mammal, e.g., a human.

Compounds of the invention may be made as described below, withmodifications to the procedure below within the skill of those ofordinary skill in the art.

EXAMPLE 1 Synthesis of the 7-Substituted Tetracycline Compounds

7-iodosancycline

The following exemplary method allows the preparation of tetracyclinecompounds of the invention having a desired purity, e.g., such that thedesired compound is provided substantially free of positional isomers.One gram of sancycline was dissolved in 25 mL of trifluoroacetic acidthat was cooled to 0° C. (on ice). 1.2 equivalents of N-iodosuccinimide(NIS) was added to the reaction mixture and reacted for forty minutes.The reaction was removed from the ice bath and was allowed to react atroom temperature for an additional five hours. The mixture was thenanalyzed by HPLC and TLC, was driven to completion by the stepwiseaddition of NIS. After completion of the reaction, the TFA was removedin vacuo and 3 mL of MeOH was added to dissolve the residue. Themethanolic solution was then added slowly to a rapidly stirring solutionof diethyl ether to form a greenish brown precipitate. The 7-iodo isomerof sancycline is purified by treating the 7-iodo product with activatedcharcoal, filtering through Celite, and subsequent removal of thesolvent in vacuo to produce the 7-isomer compound as a pure yellow solidin 75% yield.

MS (M+H) (formic acid solvent): 541.3

Rt: Hypersil C18 BDS Column, 11.73

¹H NMR: 300 MHz (Methanol d₄, TMS): δ 7.87-7.90 (d, 1H); 6.66-6.69 (d,1H); 4.06 (s, 1H); 2.98 (s, 6H); 2.42 (m, 1H); 2.19 (m, 1H); 1.62 (m,4H); 0.99 (m, 2H).

7-(3-nitrophenyl) sancycline

7-iodosancycline, 200 mg (0.28 mM), Pd(OAc)₂ 8.3 mg, and 10 mL of MeOHare added to a flask with a stir bar and the system degassed 3× usingargon. Na₂CO₃ (117 mg) dissolved in water and argon degassed is addedvia syringe is added along with 3-nitrophenylboronic acid (75 mg) inMeOH that was also degassed. The reaction was followed by HPLC for 1hour and cooled to room temperature. The solution was filtered, anddried to produce a crude mixture. The solid was dissolved indimethylformamide and injected onto a preparative HPLC system using C18reverse-phase silica. The fraction at 48-50 minutes was isolated, andthe solvent removed in vacuo to yield the product plus salts. The saltswere removed by extraction into 50:25:25 water:butanol:Ethyl acetate anddried in vacuo. This solid was dissolved in MeOH and the HCl salt madeby bubbling in HCl gas. The solvent was removed to produce the product,substantially free of positional isomers, in 42% yield as a yellowsolid.

7-(3-nitrophenyl) sancycline: Rt 16.3 min: MS (M+H, formic acidsolvent): 536.4

¹H NMR (Methanol d₄-300 MHz)δ 8.1-8.2 (m, 2H), 7.10-7.15 (m, 1H), 7.68,(m, 4H), 4.05 (s, 1H), 3.79 (s, 2H), 3.05 (s, 6H), 2.43 (m, 2H), 1.61(m, 4H), 1.05 (m, 2H)

7-(3-aminophenyl) sancycline

7-iodosancycline, 200 mg (0.37 mM), Pd(OAc)₂ 8.3 mg, and 10 mL of MeOHare added to a flask with a stir bar and the system degassed 3× usingargon. Triethylamine (103 L) is dispensed into the reaction flask viasyringe and argon degassed. The 4-aminophenyl boronic acid (128 mg)dissolved in degassed DMF (2 mL) is added via syringe. The reaction wasfollowed by HPLC for 18 hours and cooled to room temperature. Thesolution was filtered, and dried to produce a crude mixture. The solidwas dissolved in dimethylformamide and injected onto a preparative HPLCsystem using C18 reverse-phase silica. The fraction at 14.7 minutes wasisolated, and the solvent removed in vacuo to yield the product plussalts. The salts were removed by extraction into 50:25:25water:butanol:ethyl acetate and dried in vacuo. This solid was dissolvedin MeOH and the HCl salt made by bubbling in HCl gas. The solvent wasremoved to produce the product, substantially free of positionalisomers, in low yield as a yellow solid.

7-(3-aminophenyl) sancycline: Rt 4.3 min: MS (M+H, formic acid solvent):506.2

7-(4-N,N-dimethylaminophenyl) sancycline

7-iodosancycline, 600 mg (0.92 mM), Pd(OAc)₂ 30 mg, and 20 mL of MeOHare added to a flask with a stir bar and the system degassed 3× usingargon. Triethylamine (300 μl) was added via syringe is added along with4-N,N-dimethylamino-phenylboronic acid (303 mg, 1.83 mM) in MeOH thatwas also degassed. The reaction was followed by HPLC for 2 hours andcooled to room temperature. The solution was filtered, and dried toproduce a crude mixture The solid was dissolved in dimethylformamide andinjected onto a preparative HPLC system using C18 reverse-phase silica.The fraction at 17 minutes was isolated, and the solvent removed invacuo to yield the product plus salts. The salts were removed byextraction into 50:25:25 water, butanol, ethyl acetate and dried invacuo. This solid was dissolved in MeOH and the HCl salt made bybubbling in HCl gas. The solvent was removed to produce the product,substantially free of positional isomers, in 57% yield as a yellowsolid.

7-(4-N,N-dimethylaminophenyl) sancycline: Rt 7.55 min: MS (M+H, formicacid solvent): 534.2

¹H NMR (Methanol d₄-300 MHz)δ 7.02-7.05 (d, 1H), 7.79-7.82 (d, 1H),7.49-7.63 (m, 4H), 4.10 (s, 1H), 3.25-3.40 (br m, 12H), 2.58-2.70 (m,2H), 1.61(m, 4H), 1.00 (m, 2H).

7-phenylsancycline

7-iodosancycline, 150 mg (0.28 mM), Pd(OAc)₂ and 10 mL of MeOH are addedto a flask with a stir bar and the system degassed 3× using argon.Na₂CO₃ (87 mg, 0.8 mM) dissolved in water and argon degassed is addedvia syringe is added along with phenylboronic acid (68 mg, 0.55 mM) inMeOH that was also degassed. The reaction was followed by HPLC for 2hours and cooled to room temperature. The solution was filtered, anddried to produce a crude mixture. The solid was dissolved indimethylformamide and injected onto a preparative HPLC system using C18reverse-phase silica. The fraction at 36-38 minutes was isolated, andthe solvent removed in vacuo to yield the product plus salts. The saltswere removed before extraction into 50:25:25 water:butanol:ethyl acetateand dried in vacuo. This solid was dissolved in MeOH and the HCl saltmade by bubbling in HCl gas. The solvent was removed to produce theproduct, substantially free of positional isomers, in 42% yield as ayellow solid.

7-phenyl sancycline: Rt 21.6 min: MS (M+H, formic acid solvent): 491.3

¹H NMR (Methanol d₄-300 MHz)δ 7.87 (d, J=8.86 Hz, 1H), 7.38 (m, 5H),6.64 (d, 8.87 Hz, 1H), 4.00 (s, 1H), 3.84 (s, 2H), 3.01 (s, 6H), 2.46(m, 2H), 1.63 (m, 4H), 0.95 (m, 2H)

7-(4-chlorophenyl)sancycline

7-iodosancycline, 500 mg (0.91 mM), Pd(OAc)₂ 21 mg, and 20 mL of MeOHare added to a flask with a stir bar and the system degassed 3× usingargon. Na₂CO₃ (293 mg, 2.8 mM) dissolved in water and argon degassed, isadded via syringe is added along with 4-Cl-phenylboronic acid (289 mg,1.85 mM) in MeOH that was also degassed. The reaction was followed byHPLC for 45 minutes and cooled to room temperature. The solution wasfiltered, and dried to produce a crude mixture. The solid was dissolvedin dimethylformamide and injected onto a preparative HPLC system usingC18 reverse-phase silica. The fraction at 39 minutes was isolated, andthe solvent removed in vacuo to yield the product plus salts. The saltswere removed by extraction into 50:25:25 water:butanol:ethyl acetate,and dried in vacuo. This solid was dissolved in MeOH and the HCl saltmade by bubbling in HCl gas. The solvent was removed to produce theproduct, substantially free of positional isomers, in 57% yield as ayellow solid.

7-(4-chlorophenyl)sancycline: Rt 20.3 min: MS (M+H, formic acidsolvent): 525.7

¹H NMR (Methanol d₄-300 MHz)δ 7.49-7.52 (d, J=8.54 Hz, 1H), 6.99-7.01(d, 8.61 Hz, 1H), 4.12 (s, 1H), 3.67 (m, 1H), 3.06 (s, 6H), 2.58 (m,2H), 1.62(m, 4H), 1.01 (m, 2H)

7-(4-fluorophenyl)sancycline

7-iodosancycline, 200 mg (0.3 mM), Pd(OAc)₂ 8.3 mg, and 10 mL of MeOHare added to a flask with a stir bar and the system degassed 3× usingargon. Na₂CO₃ (104 mg, 1.1 mM) dissolved in water and argon degassed isadded via syringe is added along with 4-F-phenylboronic acid (104 mg,0.7 mM) in MeOH that was also degassed. The reaction was followed byHPLC for 20 minutes and cooled to room temperature. The solution wasfiltered, and dried to produce a crude mixture. The solid was dissolvedin dimethylformamide and injected onto a preparative HPLC system usingC18 reverse-phase silica. The fraction at 19-20 minutes was isolated,and the solvent removed in vacuo to yield the product plus salts. Thesalts were removed by extraction into 50:25:25 water:butanol:ethylacetate and dried in vacuo. This solid was dissolved in MeOH and the HClsalt made by bubbling in HCl gas. The solvent was removed to produce theproduct, substantially free of positional isomers, in 47% yield as ayellow solid.

7-(4-fluorophenyl)sancycline: Rt 19.5 min: MS (M+H, formic acidsolvent): 509.4

¹H NMR (Methanol d₄-300 MHz)δ 6.92-6.95 (d, 1H), 7.45-7.48 (d, 1H),7.15-7.35 (m, 4H), 4.05 (s, 1H), 3.62 (m, 1H), 3.08 (s, 6H), 2.55 (m,2H), 1.65(m, 4H), 1.00 (m, 2H)

EXAMPLE 2 In Vitro Minimum Inhibitory Concentration (MIC) Assay

The following assay is used to determine the efficacy of tetracyclinecompounds against common bacteria. 2 mg of each compound is dissolved in100 μL of DMSO. The solution is then added to cation-adjusted MuellerHinton broth (CAMHB), which results in a final compound concentration of200 μg per ml. The tetracycline compound solutions are diluted to 50 μLvolumes, with a test compound concentration of 0.098 μg/ml. Opticaldensity (OD) determinations are made from fresh log-phase broth culturesof the test strains. Dilutions are made to achieve a final cell densityof 1×10⁶ CFU/ml. At OD=1, cell densities for different genera should beapproximately: E. coli 1 × 10⁹ CFU/ml S. aureus 5 × 10⁸ CFU/mlEnterococcus sp. 2.5 × 10⁹ CFU/ml  

50 μl of the cell suspensions are added to each well of microtiterplates. The final cell density should be approximately 5×10⁵ CFU/ml.These plates are incubated at 35° C. in an ambient air incubator forapproximately 18 hr. The plates are read with a microplate reader andare visually inspected when necessary. The MIC is defined as the lowestconcentration of the tetracycline compound that inhibits growth.Compounds of the invention indicate good inhibition of growth.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures described herein. Such equivalents are considered tobe within the scope of the present invention and are covered by thefollowing claims. The contents of all references, patents, and patentapplications cited throughout this application are hereby incorporatedby reference. The appropriate components, processes, and methods ofthose patents, applications and other documents may be selected for thepresent invention and embodiments thereof.

1. A 7-substituted tetracycline compound which is substantially free ofpositional isomers, said compound having the formula:.

wherein: R⁴ and R^(4′) are each alkyl; R⁵ is hydrogen, hydroxyl, or aprodrug moiety; R⁶ and R^(6′) are each independently hydrogen, hydroxyl,alkyl, or taken together, alkenyl; R⁷ is halo substituted, N-substitutedor unsubstituted phenyl, and pharmaceutically acceptable salts thereof,wherein said tetracycline compound is substantially free of positionalisomers.
 2. The compound of claim 1, wherein R⁵, R⁶ and R^(6′) are eachhydrogen and R⁴ and R^(4′) are each methyl.
 3. The compound of claim 1,wherein R⁷ is unsubstituted phenyl.
 4. The compound of claim 1, whereinR⁷ is 2-substituted phenyl.
 5. The compound of claim 4, wherein saidcompound is selected from the group consisting of 7-(2-fluorophenyl)sancycline, 7-(2-chlorophenyl) sancycline, 7-(2-bromophenyl) sancycline,and 7-(2-iodophenyl) sancycline.
 6. The compound of claim 1, wherein R⁷is 3-substituted phenyl.
 7. The compound of claim 6, wherein saidcompound is selected from the group consisting of 7-(3-fluorophenyl)sancycline, 7-(3-chlorophenyl) sancycline, 7-(3-bromophenyl) sancycline,and 7-(3-iodophenyl) sancycline.
 8. The compound of claim 1, wherein R⁷is 4-substituted phenyl.
 9. The compound of claim 8, wherein saidcompound is selected from the group consisting of 7-(4-fluorophenyl)sancycline, 7-(4-chlorophenyl) sancycline, 7-(4-bromophenyl) sancycline,7-(4-iodophenyl) sancycline, 7-(4-trichloromethylphenyl) sancycline,7-(4-trifluoromethylphenyl) sancycline, 7-(4-tribromomethylphenyl)sancycline, and 7-(4-triiodomethylphenyl) sancycline.
 10. The compoundof claim 1, wherein R⁷ is 2-N-substituted phenyl.
 11. The compound ofclaim 10, wherein said 2-N-substituted phenyl is substituted with anitro group.
 12. The compound of claim 11, wherein said compound is7-(2-nitrophenyl) sancycline.
 13. The compound of claim 10, wherein said2-N-substituted phenyl is 2-amino substituted.
 14. The compound of claim13, wherein said 2-amino substituent is dialkylamino.
 15. The compoundof claim 14, wherein said dialkyl amino group is dimethylamino.
 16. Thecompound of claim 13, wherein said compound is selected from the groupconsisting of 7-(2-aminophenyl) sancycline,7-(2-N,N,-dimethylaminophenyl) sancycline, 7-(2-N,N,-diethylaminophenyl)sancycline, 7-(2-N,N,-dipropylaminophenyl) sancycline, and7-(2-N,N,-dibutylaminophenyl) sancycline.
 17. The compound of claim 1,wherein R⁷ is 3-N-substituted phenyl.
 18. The compound of claim 17,wherein said 3-N-substituted phenyl is substituted with a nitro group.19. The compound of claim 18, wherein said compound is 7-(3-nitrophenyl)sancycline.
 20. The compound of claim 17, wherein said 3-N-substitutedphenyl is 3-amino substituted.
 21. The compound of claim 20, whereinsaid 3-amino substituent is dialkylamino.
 22. The compound of claim 21,wherein said dialkyl amino group is dimethylamino.
 23. The compound ofclaim 20, wherein said compound is selected from the group consisting of7-(3-aminophenyl) sancycline, 7-(3-N,N,-dimethylaminophenyl) sancycline,7-(3-N,N,-diethylaminophenyl) sancycline, 7-(3-N,N,-dipropylaminophenyl)sancycline, and 7-(3-N,N,-dibutylaminophenyl) sancycline.
 24. Thecompound of claim 1, wherein R⁷ is 4-N-substituted phenyl.
 25. Thecompound of claim 24, wherein said 4-N-substituted phenyl is substitutedwith a nitro group.
 26. The compound of claim 25, wherein said compoundis 7-(4-nitrophenyl) sancycline.
 27. The compound of claim 24, whereinsaid 4-substituted phenyl is 4-amino substituted.
 28. The compound ofclaim 27, wherein said 4-amino substituent is dialkyl.
 29. The compoundof claim 28, wherein said dialkyl amino group is dimethyl.
 30. Thecompound of claim 29, wherein said compound is 7-(4-aminophenyl)sancycline, 7-(4-N,N,-dimethylaminophenyl) sancycline,7-(4-N,N,-diethylaminophenyl) sancycline, 7-(4-N,N,-dipropylaminophenyl)sancycline, or 7-(4-N,N,-dibutylaminophenyl) sancycline.
 31. Atetracycline compound which is substantially free of positional isomers,wherein said tetracycline compound is 7,9-diphenyl sancycline, andpharmaceutically acceptable salts thereof, wherein said tetracyclinecompound is substantially free of positional isomers.
 32. The compoundof any one of claims 1 or 31, wherein said compound is at least 75% freeof positional isomers.
 33. The compounds of claim 32, wherein saidcompound is at least 80% free of positional isomers.
 34. The compoundsof claim 33, wherein said compound is at least 85% free of positionalisomers.
 35. The compound of claim 34, wherein said compound is at least90% free of positional isomers.
 36. The compound of claim 35, whereinsaid compound is at least 95% free of positional isomers.
 37. A methodfor treating a tetracycline responsive state in a mammal, comprisingadministering to said mammal a 7-substituted tetracycline compound,which is substantially free of positional isomers, of formula (I):

wherein: R⁴ and R^(4′) are each alkyl; R⁵ is hydrogen, hydroxyl, or aprodrug moiety; R⁶ and R^(6′) are each independently hydrogen, hydroxyl,alkyl, or taken together, alkenyl; R⁷ is halo substituted, N-substitutedor unsubstituted phenyl; and pharmaceutically acceptable salts thereof,such that the tetracycline responsive state is treated, wherein saidtetracycline compound is substantially free of positional isomers. 38.The method of claim 37, wherein R⁵, R⁶ and R^(6′) are each hydrogen andR⁴ and R^(4′) are each methyl.
 39. The method of claim 38, wherein R⁷ isunsubstituted phenyl.
 40. The method of claim 38, wherein R⁷ is2-substituted phenyl.
 41. The method of claim 40, wherein said compoundis selected from the group consisting of 7-(2-fluorophenyl) sancycline,7-(2-chlorophenyl) sancycline, 7-(2-bromophenyl) sancycline,7-(2-iodophenyl) sancycline, 7-(2-nitrophenyl) sancycline,7-(2-aminophenyl) sancycline, 7-(2-N,N,-dimethylaminophenyl) sancycline,7-(2-N,N,-diethylaminophenyl) sancycline, 7-(2-N,N,-dipropylaminophenyl)sancycline, and 7-(2-N,N,-dibutylaminophenyl) sancycline.
 42. The methodof claim 38, wherein R⁷ is 3-substituted phenyl.
 43. The method of claim42, wherein said compound is selected from the group consisting of7-(3-fluorophenyl) sancycline, 7-(3-chlorophenyl) sancycline,7-(3-bromophenyl) sancycline, 7-(3-iodophenyl) sancycline,7-(3-nitrophenyl) sancycline, 7-(3-aminophenyl) sancycline,7-(3-N,N,-dimethylaminophenyl) sancycline, 7-(3-N,N,-diethylaminophenyl)sancycline, 7-(3-N,N,-dipropylaminophenyl) sancycline, and7-(3-N,N,-dibutylaminophenyl) sancycline.
 44. The method of claim 38,wherein R⁷ is 4-substituted phenyl.
 45. The method of claim 44, whereinsaid compound is selected from the group consisting of7-(4-fluorophenyl) sancycline, 7-(4-chlorophenyl) sancycline,7-(4-bromophenyl) sancycline, 7-(4-iodophenyl) sancycline,7-(4-trichloromethylphenyl) sancycline, 7-(4-trifluoromethylphenyl)sancycline, 7-(4-tribromomethylphenyl) sancycline,7-(4-triiodomethylphenyl) sancycline, 7-(4-nitrophenyl) sancycline,7-(4-aminophenyl) sancycline, 7-(4-N,N,-dimethylaminophenyl) sancycline,7-(4-N,N,-diethylaminophenyl) sancycline, 7-(4-N,N,-dipropylaminophenyl)sancycline, and 7-(4-N,N,-dibutylaminophenyl) sancycline.
 46. A methodfor treating a tetracycline responsive state in a mammal, comprisingadministering to said mammal 7,9-diphenyl sancycline andpharmaceutically acceptable salts thereof, which is substantially freeof positional isomers, such that the tetracycline responsive state istreated, wherein said 7,9-diphenyl sancycline is substantially free ofpositional isomers.
 47. The method of claim 37 or 46, wherein saidtetracycline responsive state is a bacterial infection.
 48. The methodof claim 47, wherein said bacterial infection is associated with E.coli.
 49. The method of claim 47, wherein said bacterial infection isassociated with S. aureus.
 50. The method of claim 47, wherein saidbacterial infection is associated with E. faecalis.
 51. The method ofclaim 47, wherein said bacterial infection is resistant to othertetracycline antibiotics.
 52. The method of claim 37 or 46, wherein saidcompound is administered with a pharmaceutically acceptable carrier. 53.The method of any one of claims 37 or 46, wherein said compound is atleast 75% free of positional isomers.
 54. The method of claim 53,wherein said compound is at least 80% free of positional isomers. 55.The method of claim 54, wherein said compound is at least 85% free ofpositional isomers.
 56. The method of claim 55, wherein said compound isat least 90% free of positional isomers.
 57. The method of claim 56,wherein said compound is at least 95% free of positional isomers.
 58. Apharmaceutical composition comprising a therapeutically effective amountof a compound of claim 1 or 31, and a pharmaceutically acceptablecarrier.
 59. The pharmaceutical composition of claim 58, wherein saidcompound is selected from the group consisting of 7-phenyl sancycline,7,9 diphenylsancycline, 7-(2-fluorophenyl) sancycline,7-(2-chlorophenyl) sancycline, 7-(2-bromophenyl) sancycline,7-(2-iodophenyl) sancycline, 7-(3-fluorophenyl) sancycline,7-(3-chlorophenyl) sancycline, 7-(3-bromophenyl) sancycline,7-(3-iodophenyl) sancycline, 7-(4-fluorophenyl) sancycline,7-(4-chlorophenyl) sancycline, 7-(4-bromophenyl) sancycline,7-(4-iodophenyl) sancycline, 7-(4-trichloromethylphenyl) sancycline,7-(4-trifluoromethylphenyl) sancycline, 7-(4-tribromomethylphenyl)sancycline, 7-(4-triiodomethylphenyl) sancycline, 7-(2-nitrophenyl)sancycline, 7-(2-aminophenyl) sancycline, 7-(2-N,N,-dimethylaminophenyl)sancycline, 7-(2-N,N,-diethylaminophenyl) sancycline,7-(2-N,N,-dipropylaminophenyl) sancycline, 7-(2-N,N,-dibutylaminophenyl)sancycline, 7-(3-nitrophenyl) sancycline, 7-(3-aminophenyl) sancycline,7-(3-N,N,-dimethylaminophenyl) sancycline, 7-(3-N,N,-diethylaminophenyl)sancycline, 7-(3-N,N,-dipropylaminophenyl) sancycline,7-(3-N,N,-dibutylaminophenyl) sancycline, 7-(4-nitrophenyl) sancycline,7-(4-aminophenyl) sancycline, 7-(4-N,N,-dimethylaminophenyl) sancycline,7-(4-N,N,-diethylaminophenyl) sancycline, 7-(4-N,N,-dipropylaminophenyl)sancycline, and 7-(4-N,N,-dibutylaminophenyl) sancycline.
 60. Thepharmaceutical composition of claim 58 wherein said compound is at least75% free of positional isomers.
 61. The pharmaceutical composition ofclaim 60, wherein said compound is at least 80% free of positionalisomers.
 62. The pharmaceutical composition of claim 61, wherein saidcompound is at least 85% free of positional isomers.
 63. Thepharmaceutical composition of claim 62, wherein said compound is atleast 90% free of positional isomers.
 64. The pharmaceutical compositionof claim 63, wherein said compound is at least 95% free of positionalisomers.